PROJECT SUMMARY The University of Pittsburgh Drug Discovery Institute (UPDDI) is requesting funds to purchase the Perkin Elmer OPERA PHENIX high speed, high resolution spinning disk confocal High-Content Screening (HCS) device. The Opera Phenix will replace two Molecular Devices ImageXpress Ultra high content readers purchased in 2008, which are critical to multiple NIH-, DoD-, and Foundation-funded projects at the University of Pittsburgh, but are no longer supported by the manufacturer and have been decommissioned. We have determined that one Opera Phenix instrument can replace the two IXUs. The Phenix is a third generation HCS instrument that will be essential to satisfy the diverse needs of users that the UPDDI serves. No comparable instruments exist at the University of Pittsburgh, the University of Pittsburgh Medical Center, and Carnegie Mellon University. Over the last decade, HCS has become a standard in the pharmaceutical industry for target identification, phenotypic screening, as well as toxicology, and in academia for large-scale biological studies, where cell-by- cell quantitation is critical. The UPDDI has been an academic pioneer in the application of HCS and serves an extensive number of collaborators across campus that require and rely on HCS, ranging from neurodegeneration, organ regeneration, cancer, liver diseases, organotypic model development, and traumatic brain injury. Our diverse user groups? needs emphasize discovery models of physiological relevance and high complexity, and therefore require fast, high resolution 2D, 3D, and kinetic imaging and maximum flexibility in image analysis. The large number of HCS users working in the UPDDI further demands a fast system to permit effective sharing of instrument time, and an integrated database with off-site user access to perform off- line analysis. Key requirements for an HCS imager therefore are superior speed in acquiring z-series of images at high resolution of thick specimens in aqueous matrices, mature yet flexible image algorithms, and seamless integration of instrument software with system, public,and custom-developed UPDDI databases. The only instrument that meets all of these criteria is the Opera Phenix because it has 1) fast laser-based illumination and the ability to acquire multiple channels simultaneously 2) water immersion objectives that eliminate non-matching refractive indices, which limit spherical aberrations of air and oil objectives at longer working distances and require adjustment of correction collars depending on imaging depth; 3) a powerful suite of user-friendly yet flexible image analysis routines including a 3D module, advanced texture and morphology analysis, and intuitive and user-friendly machine learning; and 4) the ability to perform seamless ?adaptive high-resolution imaging?, i.e., pre-scanning a large area at low magnification, followed by automated ?on-the- fly? switching to higher magnification to acquire high resolution images of user-defined regions of interest. The Opera Phenix is the only instrument on the market that is capable of fulfilling the demands of the University of Pittsburgh?s diverse drug discovery community.